Cryptococcus neoformans is a human fungal pathogen that can progress to systemic infection in both immunocompromised and healthy individuals, leading to lethal cryptococcal meningitis. Current therapies to address C. neoformans infection are not highly effective, therefore more efficacious treatments are needed to prevent infection, particularly in vulnerable patient population such as organ transplant recipients, HIV-AIDS patients and cancer patients undergoing chemotherapy. Copper (Cu) serves as an essential co-factor for a wide variety of enzymes in fungi and humans, but excess Cu is toxic requiring organisms to maintain tight control of intracellular Cu levels. Studies suggest that the C. neoformans Cu-sensing transcription factor, Cuf1, is required for virulence in mouse infection models. While Cuf1 activates the expression of genes encoding proteins that carry out Cu import, recent work in the laboratory of my postdoctoral sponsor, Dennis J. Thiele, has demonstrated that Cuf1 activates many genes that are essential for both Cu acquisition and Cu detoxification, as well as genes encoding proteins of unknown function. Moreover, work by others suggests that activated macrophages accumulate Cu within the lumen of the phagosome as an anti-microbial agent. Since alveolar macrophages are the first line of defense against C. neoformans infection, it is important to understand how the Cuf1 Cu-sensing transcription factor, and the proteins encoded by Cuf1 target genes, play a critical role in C. neoformans virulence. In this application I outline experiments to accomplish this overall goal through three Specific Aims. In the first aim I will carry out genetic and biochemical experiments to establish whether the genes that are dependent on Cuf1 are regulated directly or indirectly by this transcription factor and to ascertain the role of these genes in Cu acquisition or detoxification. In the second aim I outline experiments to evaluate mutants in Cuf1-dependent genes to ascertain their contribution to survival to alveolar macrophage phagocytosis in vitro. In the third aim, using both wild type mice and mice created in my postdoctoral sponsor's laboratory that are specifically defective in macrophage Cu accumulation, I outline experiments to decipher what roles Cuf1-dependent genes and the host Cu homeostasis machinery play in host-pathogen interactions. The new expertise I will gain by carrying out this project at the intersection of fungal pathogenesis, Cu metalloregulation and the genetics of mammalian host Cu metabolism will contribute significantly to understanding the role of Cu in the host-pathogen axis and will provide me with cutting-edge training to launch a career as a competitive independent faculty member. PUBLIC HEALTH RELEVANCE: Cryptococcus neoformans is a fungal pathogen that infects both immunocompromised and healthy individuals and if left untreated, C. neoformans can result in lethal fungal meningitis. Recently, it has become clear that copper plays a critical rolein the outcome of infection, with the host using the toxic properties of copper and pathogens encoding copper defense mechanisms. In this application I outline experiments to characterize the C. neoformans copper metabolism mechanisms in the context of infection with the aim of identifying novel and more efficacious drug targets against this deadly pathogen.